RESEARCH ARTICLE

Analysis of immune cell populations in atrial

myocardium of patients with atrial fibrillation

or sinus rhythm

Natalia Smorodinova1, Martin Blaha2, Vojtěch Melenovsky2, Karolına Rozsıvalova1,

Jaromır Přidal1, Maria Durisova1, Jan Pirk3, Josef Kautzner2, Tomas Kučera1*

1 Institute of Histology and Embryology, The First Faculty of Medicine, Charles University, Prague, Czech

Republic, 2 Institute for Clinical and Experimental Medicine-IKEM, Department of Cardiology, Prague, Czech

Republic, 3 Institute for Clinical and Experimental Medicine-IKEM, Department of Cardiovascular Surgery,

Prague, Czech Republic

* tkucer@lf1.cuni.cz

Abstract

Background

Atrial fibrillation (AF) is the most common arrhythmia and despite obvious clinical impor-

tance remains its pathogenesis only partially explained. A relation between inflammation

and AF has been suggested by findings of increased inflammatory markers in AF patients.

Objective

The goal of this study was to characterize morphologically and functionally CD45-positive

inflammatory cell populations in atrial myocardium of patients with AF as compared to sinus

rhythm (SR).

Methods

We examined 46 subjects (19 with AF, and 27 in SR) undergoing coronary bypass or valve

surgery. Peroperative bioptic samples of the left and the right atrial tissue were examined

using immunohistochemistry.

Results

The number of CD3+ T-lymphocytes and CD68-KP1+ cells were elevated in the left atrial

myocardium of patients with AF compared to those in SR. Immune cell infiltration of LA was

related to the rhythm, but not to age, body size, LA size, mitral regurgitation grade, type of

surgery, systemic markers of inflammation or presence of diabetes or hypertension. Most of

CD68-KP1+ cells corresponded to dendritic cell population based on their morphology

and immunoreactivity for DC-SIGN. The numbers of mast cells and CD20+ B-lymphocytes

did not differ between AF and SR patients. No foci of inflammation were detected in any

sample.

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 1 / 13

a1111111111

a1111111111

a1111111111

a1111111111

a1111111111

OPENACCESS

Citation: Smorodinova N, Blaha M, Melenovsky V,

Rozsıvalova K, Přidal J, Durisova M, et al. (2017)

Analysis of immune cell populations in atrial

myocardium of patients with atrial fibrillation or

sinus rhythm. PLoS ONE 12(2): e0172691.

doi:10.1371/journal.pone.0172691

Editor: Maria Cristina Vinci, Centro Cardiologico

Monzino, ITALY

Received: October 20, 2016

Accepted: February 8, 2017

Published: February 22, 2017

Copyright: © 2017 Smorodinova et al. This is an

open access article distributed under the terms of

the Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information

files.

Funding: This work was supported by the

Research Program of Charles University –

PRVOUK P25/LF1/2 (TK), www.cuni.cz, Ministry of

Health for Development of Research Organization

00023001, http://www.mzcr.cz/ (IKEM, institutional

support) (JK), and by the EU Operational Program

Prague—Competitiveness: project CEVKOON

(CZ.2.16/3.1.00/22126), http://www.ikem.cz/cs/

Conclusions

An immunohistochemical analysis of samples from patients undergoing open heart surgery

showed moderate and site-specific increase of inflammatory cells in the atrial myocardium

of patients with AF compared to those in SR, with prevailing population of monocyte-macro-

phage lineage. These cells and their cytokine products may play a role in atrial remodeling

and AF persistence.

Introduction

From all cardiac arrhythmias in human, atrial fibrillation (AF) is one of the most common and

important irregularities of the heart rhythm. The prevalence of AF is 1–2% in general popula-

tion, but it reaches up to 10% in elderly subjects [1]. It confers approximately 5-fold higher

risk of stroke; one in five of all strokes is attributed to this arrhythmia and the strokes associ-

ated with AF are more severe than strokes from other etiologies [2]. Despite its obvious clinical

importance, the pathogenesis of AF remains only partially explained. The current classification

of AF is arbitrary and based mainly on its duration [3]. Paroxysmal AF is defined as recurrent

AF that terminates spontaneously within several days. Persistent AF is defined as AF which is

sustained beyond seven days, or lasting less than seven days but necessitating pharmacologic

or electrical cardioversion. Included within the category of persistent AF is “longstanding per-

sistent AF” which is defined as continuous AF of greater than one-year duration. The term

permanent AF is defined as AF in which cardioversion has either failed or not been attempted,

and arrhythmia is accepted as permanent rhythm. Unfortunately, this classification does not

take into account the real underlying atrial substrate and there has been a call for a mechanistic

classification of AF with important therapeutic benefits [4]. The main factors predisposing to

AF are aging, arterial hypertension, valvular disease, congestive heart failure and coronary

artery disease [5]. Together with diabetes mellitus, lung disease, peri-and myocarditis, cardio-

myopathy and thyroid disease these predisposing factors belong to well-known conditions

capable of favoring this arrhythmia [5]. However, new predisposing factors are also emerging,

such as obstructive sleep apnea, obesity, heavy alcohol consumption, endurance sport activities

and gene mutations [5–7]. These risk factors lead to functional and morphological changes

that can support maintenance of AF and are believed to be responsible for a progressive char-

acter of the arrhythmia that tends to develop over time from paroxysmal to permanent form.

Three forms of atrial remodeling during a progression of AF have been described: electrical,

contractile and structural [8]. Electrical remodeling is a consequence of high atrial rate and

includes shortening of the refractory period of atrial cardiomyocytes and slowing the velocity

of atrial conduction [9]. The structural remodeling is characterized both by changes in cardio-

myocytes [10, 11] and in the endomysium [12, 13], by changes in extracellular matrix compo-

sition and atrial fibrosis [14–16]. The changes at the level of cardiomyocytes include the loss of

contractile structures (“myocytolysis”), a switch to more fetal-like phenotype as manifested by

altered expression of certain proteins, accumulation of glycogen and other changes at the ultra-

structural level [6]. Changes in the interstitium are mainly manifested by the deposition of col-

lagen fibers around cardiomyocytes [17]. Contractile remodeling is caused mainly by impaired

calcium handling and may result in atrial mechanical dysfunction that may be transient (stun-

ning). Impaired contractility can result not only from the changes of cell physiology, but also

structural remodeling at the level of individual cardiomyocytes as well as at the level of the

whole atrial tissue. The three forms of the atrial remodeling are thus interrelated [8]. Another

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 2 / 13

centrum-exp-mediciny/cevkoon/a-1676/ (JK, VM).

The funders had no role in study design, data

collection and analysis, decision to publish, or

preparation of the manuscript.

Competing interests: The authors have declared

that no competing interests exist.

morphological feature related to AF is the presence of inflammatory cells in the atrial myocar-

dium [11]. Despite observed association between elevated plasma levels of inflammatory mark-

ers and AF, it is still not determined whether inflammation is a systemic or local phenomenon

reflecting an active inflammatory process in the heart [18]. A role of inflammation and myo-

cardial inflammatory infiltrate was suggested by morphological studies and by clinical studies

that monitored serum levels of inflammatory cytokines in patients with AF [18, 19]. Histologi-

cal findings supporting the association between inflammation and AF have been reported in

several animal [20, 21] as well as human studies [11, 22–24]. Other reports have shown a corre-

lation between the level of fibrosis and infiltration of atrial myocardium by inflammatory cells

[25]. However, the above studies focused on inflammatory cells in general and were usually

limited to one atrium only or to single pathological condition related to AF. It is not known

whether the inflammatory cells are a marker of local reaction to tissue injury caused by factors

leading to AF or whether they actively participate in the maintenance of AF due to direct cyto-

toxic or pro-fibrotic effects [26] or indirectly due to released cytokines that may promote

arrhythmogenesis [21]. The aims of this study were the following: 1) to characterize and quan-

tify immune cell populations in human atrial myocardium of patients undergoing open heart

surgery with AF and in sinus rhythm (SR); 2) to identify regional differences in terms of

immune cell populations in atrial myocardium of patients undergoing open heart surgery; 3)

to correlate atrial immune cell populations with markers of systemic inflammation or AF risk

factors.

Materials and methods

Patients

We used bioptic material from 46 patients (19 with long-term persistent AF and 27 in SR),

which were hospitalized in Institute for Clinical and Experimental Medicine in Prague. We

included patients who underwent coronary artery bypass surgery or valve surgery and who

agreed to participate in the study (S1 Table). Patients who presented with sepsis, active endo-

carditis, permanent cardiostimulation, dominating atrial flutter or postincisional supraventric-

ular tachycardia on ECG were excluded. The study conformed to the principles presented in

the Declaration of Helsinki. Ethics committee of the Institute for Clinical and Experimental

Medicine in Prague approved the study protocol. Only those patients who signed a written

informed consent were included into the study.

Tissue sampling, histological and immunohistochemical analysis

Tissue samples were obtained during open-heart surgery and processed as described previ-

ously [27]. Immunohistochemistry was used to visualize CD45, CD3, CD68-KP1, DC-SIGN

(DC-specific ICAM-3 grabbing nonintegrin), mast cell tryptase and CD20 in these samples

(S2 Table).

Histomorphometry

To quantify CD45+cells, CD3+cells, CD68-KP1+cells and mast cells we used the program

Image J 1.44p (National Institutes of Health, USA). Image sampling was performed as de-

scribed in [27]. Only immunoreactive cells within the myocardium were counted manually

and labeled in the image analysis program to make sure that no cell is counted twice. The fre-

quency of cells was expressed as the number of cells per square millimeter. Those, who per-

formed image analysis were blinded to patient characteristics.

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 3 / 13

Statistical analysis

The values of morphometric analyses are expressed as a mean±SD. The range of values is also

provided. Statistical testing of the morphometric analysis comparing values from AF and SR

groups in various anatomical locations was performed using a non-parametric test—Mann–

Whitney U test. Statistical significance of differences between the patient groups in various

patient characteristics was tested using Student’s t-test, Fisher’s exact test, or chi-square test

where appropriate. Statistical programs used for these analyses were Microsoft Office Excel,

InStat and Statistica. A value of P< 0.05 was considered significant.

Results

Patient characteristics

S1 Table shows characteristics of the patient population in detail. The group of patients suffer-

ing from AF was 7 years older on average. Patients from AF group had higher left and right

atrial volumes. Left atrial volume was on average almost doubled in subjects with AF. On the

other hand, patients in SR had more often coronary artery disease. There was no difference in

NYHA class, diabetes, arterial hypertension, anemia and renal insufficiency between both

patient groups. E/E ‘ratio, non-invasive estimate of left atrial pressure and markers of systemic

inflammation (CRP, leukocyte count) were similar between patients with AF and SR.

Detection and quantification of CD45+ cells in the atrial myocardium

To visualize cells with a potential of an inflammatory process we used an antibody against a

pan-leukocyte marker CD45. This marker is common for cells of hematopoietic origin. Immu-

noperoxidase reaction revealed CD45+ cells in all samples analyzed from SR and AF group.

The immunoreactive cells were found in the whole thickness of the atrial wall regardless of

the anatomical location (Fig 1A–1D). In the epicardium, CD45+ cells often formed larger

clusters (Fig 1A). In the endocardium, these cells were scattered in the whole thickness of this

layer (Fig 1B), occasionally they also adhered to the endothelium from the luminal side (not

shown). The cells immunoreactive for CD45+ were found in the interstitial compartment of

the myocardium both in perimysium and in endomysium. Morphologically, the CD45+ cell

population appeared heterogeneous with cells having in general either rounded or elongated

cell bodies (Fig 1D). The CD45+ cells located in the myocardium were quantified in tissue sec-

tions of left and right atrial appendages and left atrial free wall. When samples from SR group

and AF group were compared there was a tendency for a higher frequency of CD45+ cells in

AF group, however, without reaching a statistical significance (Fig 1E). In the right atrial ap-

pendage, the average number of CD45+ cells per 1mm2 was 42.4±25.5 (range 12.2–103.8) in

the SR group and 58.4±31.3 (range 12.6–118.5) in the AF group. In the left atrial appendage,

the average number of CD45+ cells per 1mm2 was 36.1±16.7 (range 14.4–61.7) in the SR group

and 54.2±52.2 (range 17.8–186.2) in the AF group. In the left atrial free wall, the average num-

ber of CD45+ cells per 1mm2 was 36.2±14.2 (range 20.6–59.3) in the SR group and 39.0±23.7

(range 11.9–95.5) in the AF group. In the pooled samples from the whole left atrium, the aver-

age number of CD45+ cells per 1mm2 was 36.1±15.9 (range 14.3–61.7) in the SR group and

46.6±41.2 (range 11.9–186.2) in the AF group.

CD3-positive T-lymphocytes in the atrial myocardium

With the aim to characterize the inflammatory cell population further, we performed analysis

of different subpopulations of CD45+ cells. Thus, as a next step, T-lymphocytes were immu-

nohistochemically detected in atrial samples using an antibody against a marker CD3. Like

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 4 / 13

Fig 1. CD45-positive cells in the atrial myocardium. Sections of atrial wall from patients with sinus rhythm (A, B, D) and atrial

fibrillation (C) showing a result of immunoperoxidase reaction for CD45. A) Epicardial and myocardial layer of atrial wall contains

scattered as well as clustered (arrowhead) CD45-positive cells. Scale bar = 100μm. B) Endocardial layer with CD45-positive

cells (arrowhead) next to the atrial lumen (L). Scale bar = 20μm. C) A section through the trabecular part of the atrial wall with

CD45-positive cells in the myocardium (M) and endocardium (E). Scale bar = 50 μm. D) A detailed view on the atrial myocardium

with CD45-positive cells having rather rounded (arrowhead) or elongated (arrow) cell shape. Scale bar 50 = μm. E) Frequency of

CD45+ cells in the atrial myocardium of patients with atrial fibrillation (AF) and sinus rhythm (SR). An average number of CD45

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 5 / 13

CD45+ cells, T-lymphocytes were found in all samples from both SR and AF groups and were

localized in the whole atrial wall including myocardium (Fig 2), but were much less abundant

than CD45+ cells. The rounded morphology of CD3+ cells, which were found scattered in

endomysial and perimysial interstitial spaces corresponded to that of lymphocytes (Fig 2A).

The quantification of CD3+ T-lymphocytes revealed their significantly higher frequency in left

atrium of patients with AF. When samples of left atrial appendage and left atrial free wall were

pooled the difference between AF and SR group was statistically significant (Fig 2C). In the

right atrial appendage, the average number of CD3+ cells per 1mm2 was 6.5±6.0 (range 1.08–

26.8) in the SR group and 5.3±4.2 (range 1.2–15.3) in the AF group. In the left atrial append-

age, the average number of CD3+ cells per 1mm2 was 3.5±2.3 (range 1.1–8.4) in the SR group

and 8.6±10.1 (range 1.7–36.2) in the AF group. In the pooled samples from the whole left

atrium, the average number of CD3+ cells per 1mm2 was 3.5±2.2 (range 1.1–8.4) in the SR

group and 9.8±10.0 (range 1.7–36.2) in the AF group.

CD68-KP1-positive cells in the atrial myocardium

Inflammatory cells including monocyte/macrophages and dendritic cells can be detected

using a marker CD68-KP1. Cells immunoreactive for CD68-KP1 were very frequent in all

atrial tissue samples from both AF and SR group. They were regularly detected in the myocar-

dium and had predominantly an elongated cell shape (Fig 2B). The quantitative analysis

revealed that CD68-KP1+ cell population was the most abundant inflammatory cell popula-

tion and they were more frequent in samples from patients with AF (Fig 2D). There was signif-

icantly higher number of CD68-KP1+ cells in the left appendage and in pooled samples of left

appendage and left atrial wall in patients with AF compared to patients with SR (Fig 2D). In

the right atrial appendage, the average number of CD68-KP1+ cells per 1mm2 was 44.3±19.3

(range 7.7–76.7) in the SR group and 52.0±27.0 (range 14.0–109.0) in the AF group. In the left

atrial appendage, the average number of CD68-KP1+ cells per 1mm2 was 40.8±24.4 (range

9.9–96.3) in the SR group and 67.2±25.0 (r[7]ange 41.6–122.4) in the AF group. In the pooled

samples from the whole left atrium, the average number of CD68-KP1+ cells per 1mm2 was

38.5±23.7 (range 9.9–96.3) in the SR group and 60.2±28.4 (range 18.0–122.4) in the AF group.

DC-SIGN-positive dendritic cells, mast cells and B-lymphocytes in the

atrial myocardium

Dendritic cells are among cells immunoreactive for CD68-KP1+. To confirm their presence in

the atrial myocardium of patients undergoing open heart surgery we performed an immuno-

histochemical detection of marker DC-SIGN, which labels immature dendritic cells. DC-

SIGN+ cells were found frequently in atrial myocardial samples from both AF and SR groups.

They had the same morphology as the cells previously immunoreactive for CD68-KP1 antigen

(Fig 3A). Mast cells were detected using an antibody against mast cell tryptase.

Mast cells were quite rare in the atrial myocardium and they were found scattered as indi-

vidual cells in the endomysial compartment or in small groups in the perimysial spaces sur-

rounding bigger vessels (Fig 3B). There was a similar frequency of mast cells in the atrial

myocardium of patients in AF and SR group (not shown). B-lymphocytes were detected using

an antibody against CD20 marker in the atrial myocardium of patients in AF and SR group.

+ cells per square mm of cross-sectioned atrial myocardium is given +- SD. Right appendage–SR (n = 22), AF (n = 15); Left

appendage—SR (n = 8), AF (n = 8); Left atrial free wall—SR (n = 4), AF (n = 8); Left atrial free wall + left appendage—SR

(n = 12), AF (n = 16).

doi:10.1371/journal.pone.0172691.g001

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 6 / 13

CD20+ cells were occasionally found as small clusters in the epicardial layer (Fig 3C). In the

myocardium, these cells were very rare (Fig 3D).

Determinants of inflammatory cell populations other than cardiac rhythm

In the pooled cohort, variations of immune cell population were related to prevailing rhythm

(particularly for CD3 and CD68-KP1 markers). However, they did not significantly correlate

with mitral regurgitation grade, left atrial volume, E/E´ ratio, age, presence of diabetes,

Fig 2. CD3-positive T-lymphocytes and CD68-KP1-positive cells in the atrial myocardium. Sections of atrial myocardium from patients with atrial

fibrillation showing a result of immunoperoxidase reaction for CD3 and CD68-KP1. A) CD3-positive T-lymphocytes localized among atrial cardiomyocytes and

in the interstitial spaces (arrowheads). Scale bar = 50 μm. B) CD68-KP1-positive cells are found in the interstitium and have mainly elongated morphology

(arrowheads). Scale bar = 50 μm. C) Frequency of CD3-positive T-lymphocytes in the atrial myocardium of patients with atrial fibrillation (AF) and sinus rhythm

(SR). An average number of CD3-positive T-lymphocytes cells per square mm of cross-sectioned atrial myocardium is given +- SD. Right appendage–SR

(n = 17), AF (n = 11); Left appendage—SR (n = 8), AF (n = 10); Left atrial free wall + left appendage—SR (n = 9), AF (n = 16). *—p<0,05 D) Frequency of

CD68-KP1-positive cells in the atrial myocardium of patients with atrial fibrillation (AF) and sinus rhythm (SR). An average number of CD68-KP1-positive cells

per square mm of cross-sectioned atrial myocardium is given +- SD. Right appendage–SR (n = 22), AF (n = 9); Left appendage—SR (n = 11), AF (n = 9); Left

atrial free wall + left appendage—SR (n = 13), AF (n = 19). *—p<0,05

doi:10.1371/journal.pone.0172691.g002

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 7 / 13

hypertension or coronary artery disease. Inflammatory infiltrate was related to the rhythm

itself, rather than to risk factors known to be associated with AF. We also did not observe sig-

nificant correlation between atrial inflammatory infiltration by CD45+, CD3+ or CD68-KP1+

and CRP or blood leukocyte count.

Discussion

The results of this study can be summarized as follows: 1) The present study shows that CD45

+ cells are a common finding in atrial myocardium of patients undergoing open heart surgery

regardless of the actual heart rhythm; 2) An analysis of CD45+ cell count was performed in dif-

ferent anatomical locations–left atrial appendage, left atrial free wall and right atrial append-

age; 3) There was a tendency for a higher CD45+ cell count in samples from patients with AF

Fig 3. DC-SIGN-positive cells, mast cells and B-lymphocytes in the atrial wall. A) A section of atrial myocardium from a patient with atrial fibrillation

showing a result of immunoperoxidase reaction for DC-SIGN. DC-SIGN—positive cells have mainly elongated morphology (arrowheads). Scale bar = 50 μm.

B) A section of atrial myocardium from a patient with sinus rhythm. Mast cells detected using anti-mast cell tryptase antibody are scattered in the atrial

myocardium. Scale bar = 50 μm. (C-D) Sections of atrial wall from a patient with sinus rhythm. C) A cluster of CD20-positive B-lymphocytes in the epicardial

layer (arrowhead). Scale bar = 100 μm. D) A detail of atrial myocardium with an isolated CD20-positive B-lymphocyte. Scale bar = 50 μm.

doi:10.1371/journal.pone.0172691.g003

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 8 / 13

and this difference was even more prominent when focusing on some specific subpopulations

of inflammatory cells in the atrial myocardium; 4) Both in case of CD3+ T-lymphocytes and

CD68-KP1+ cells there was a higher cell count in the samples from patients with AF, but only

in the left atrium.

Several studies have been published previously describing an elevated count of inflamma-

tory cells in the atria of patients suffering from AF [11, 15, 22–25, 28, 29]. In contrast to our

study, some papers evaluated samples only from either left or right atrium [22, 24, 28, 29] or

the analysis was performed on necroptic specimens [25]. The results of our study are in general

agreement with findings of the above studies performed on different patient populations.

Importantly, it shows that there is significantly larger immune cell count only in the left atrium

of patients suffering from AF. The reason for the differential response of atria in terms of

inflammatory cell infiltration might be explained by a differences in density of capillaries serv-

ing as an entrance port for immune cells to invade atrial interstitium. In our recent study per-

formed on the same patient groups, we reported a higher microvascular density in the left

atrium compared to the right atrium [27]. It was shown repeatedly that the most pronounced

morphological changes attributable to AF could be detected in the left posterior atrial wall

[17]. Since, in the present study, most samples of the left atrium originated from the left ap-

pendage and a smaller number from left atrial free wall, we could not evaluate them separately,

however the statistical significance of differences between CD3+ and CD68-KP1+ always

increased when the samples from both locations were pooled together.

In addition to above mentioned immune cell populations, we also evaluated the amount of

mast cells and B-lymphocytes. One experimental study suggested the crucial role of mast cells

for development of AF [21]. However, in our study we found only a small number of mast cells

in the atrial myocardium and there was no difference between AF and SR groups of patients.

Our data show rather moderate increase of immune cells in left atrial myocardium of

patients with AF compared to SR group of patients undergoing open heart surgery. In case of

CD3+ T-lymphocytes, we found diffuse infiltration mostly by isolated cells. Interestingly, in

contrast to an initial report [11], no inflammatory infiltrates were found in more recent study

analyzing atrial myocardium from patients with lone atrial fibrillation [7]. In the present

study, we were not able to detect active (acute) myocarditis according to current criteria [30,

31]. It seems likely that an elevated number of CD3+ and CD68-KP1+ cells in the left atrial

myocardium of patients with AF reflects certain level of inflammatory activation of myocardial

tissue due to the mechanical stress accompanying the atrial dilation [32, 33]. Expression of

inflammatory cytokines is another parameter of local inflammatory state. One study reported

that there was an increased production of TNF-alpha and IL-6 in the right atria of AF patients

with valvular disease compared to control group of SR patients with valvular disease [34]. A

more recent study demonstrated a higher expression of an inflammatory cell adhesion mole-

cule VCAM1 in myocardial capillaries of left atrial appendages of patients with AF compared

with autoptic SR controls [29]. Interestingly, while the former study also reported a higher col-

lagen volume fraction in patients with AF [34], no difference between AF patients and controls

was observed in the latter [29]. Fibrosis is considered by many authors as a hallmark of struc-

tural remodeling in AF [13–16] and can also cause remodeling of infiltrating subepicardial

fatty infiltrates [35]. However, there are also reports, where no statistical difference in collagen

volume fraction or fibrosis was found between AR and SR group of patient most likely due to a

variable influence of accompanying structural heart diseases [27, 29, 32, 36, 37]. The largest

immune cell population was formed by CD68-KP1+ cells of monocyte/macrophage lineage

corresponding largely to dendritic cells, which were immunoreactive to DC-SIGN. The role

of these cells that are also found in the normal myocardium [38] is still a matter of ongoing

research and these cells can have both beneficial as well as detrimental effects on the heart,

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 9 / 13

based on various pathophysiological circumstances [39, 40]. In a recent study it was demon-

strated that atrial myocarditis coincides with certain types of ventricular myocarditis [41], but

since we have no reports of ventricular myocarditis in our patients we are unable to relate an

increased number of CD3+ and CD68-KP1+ cells in the left atria of AF group to an inflamma-

tory process in other parts of the myocardium. Our goal was to quantify inflammatory cells in

the atrial myocardia. Although we observed inflammatory cells regularly in the endocardium

and epicardium regardless of the heart rhythm we did not perform a quantitative study in

these layers of the heart wall and thus future studies may demonstrate whether there is a corre-

lation between the number of these cells across the heart. There are several limitations result-

ing from the fact that we relied on material from bioptic samples. In addition, the small size of

our patient groups might affect our quantitative results. It was possible to safely harvest only

small bioptic samples and these could be obtained from only certain regions within the atria.

Only patients suffering from various structural heart diseases were compared.

Conclusions

Immunohistochemical analysis of tissue samples from patients undergoing open heart surgery

showed moderate and site-specific increase of inflammatory cells in the atrial myocardium of

patients with AF compared to those in SR. The most represented immune cell population was

CD3+ T-lymphocytes and CD68-KP1+ monocyte/macrophage subpopulation corresponding

mostly to dendritic cells. Mast cells and B-lymphocytes were less frequent. Only CD3+ and

CD68-KP1+ cells were elevated in the left atrium of patients with AF, while no inflammatory

foci were detected in the atrial myocardium. This local elevation of some CD45+ cell popula-

tions might reflect the progression of AF. More research has to be done to elucidate signifi-

cance of general pro-inflammatory state reported in patients with AF.

Supporting information

S1 Table. Characteristics of patients with AF and patients in SR.

(DOCX)

S2 Table. Characterization of antibodies used in the study.

(DOC)

Acknowledgments

We thank Bc. Ivana Kamenařova for the help with sample collection, Ms. Marcela Blazkeova

and Ing.Lucie Kosova for their excellent technical assistance.

Author Contributions

Conceptualization: TK JK VM J Pirk.

Data curation: TK MB VM.

Formal analysis: TK MB VM.

Funding acquisition: TK VM JK J Pirk.

Investigation: TK NS VM MB MD J Přidal KR.

Methodology: JK VM TK.

Project administration: TK VM JK J Pirk.

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 10 / 13

Resources: TK VM JK J Pirk.

Supervision: TK JK J Pirk.

Visualization: NS TK J Přidal KR MD.

Writing – original draft: NS TK VM JK.

Writing – review & editing: NS TK VM JK.

References1. Heeringa J, van der Kuip DA, Hofman A, Kors JA, van Herpen G, Stricker BH, et al. Prevalence, inci-

dence and lifetime risk of atrial fibrillation: the Rotterdam study. Eur Heart J. 2006; 27(8):949–53. doi:

10.1093/eurheartj/ehi825 PMID: 16527828

2. European Heart Rhythm A, European Association for Cardio-Thoracic S, Camm AJ, Kirchhof P, Lip

GY, Schotten U, et al. Guidelines for the management of atrial fibrillation: the Task Force for the Man-

agement of Atrial Fibrillation of the European Society of Cardiology (ESC). Eur Heart J. 2010; 31

(19):2369–429. doi: 10.1093/eurheartj/ehq278 PMID: 20802247

3. European Heart Rhythm A, European Cardiac Arrhythmia S, American College of C, American Heart A,

Society of Thoracic S, Calkins H, et al. HRS/EHRA/ECAS expert Consensus Statement on catheter

and surgical ablation of atrial fibrillation: recommendations for personnel, policy, procedures and

follow-up. A report of the Heart Rhythm Society (HRS) Task Force on catheter and surgical ablation

of atrial fibrillation. Heart Rhythm. 2007; 4(6):816–61. doi: 10.1016/j.hrthm.2007.04.005 PMID:

17556213

4. Fabritz L, Guasch E, Antoniades C, Bardinet I, Benninger G, Betts TR, et al. Expert consensus docu-

ment: Defining the major health modifiers causing atrial fibrillation: a roadmap to underpin personalized

prevention and treatment. Nat Rev Cardiol. 2016; 13(4):230–7. doi: 10.1038/nrcardio.2015.194 PMID:

26701216

5. Wakili R, Voigt N, Kaab S, Dobrev D, Nattel S. Recent advances in the molecular pathophysiology of

atrial fibrillation. J Clin Invest. 2011; 121(8):2955–68. PubMed Central PMCID: PMCPMC3148739. doi:

10.1172/JCI46315 PMID: 21804195

6. Corradi D. Atrial fibrillation from the pathologist’s perspective. Cardiovasc Pathol. 2014; 23(2):71–84.

doi: 10.1016/j.carpath.2013.12.001 PMID: 24462196

7. Corradi D, Callegari S, Manotti L, Ferrara D, Goldoni M, Alinovi R, et al. Persistent lone atrial fibrillation:

clinicopathologic study of 19 cases. Heart Rhythm. 2014; 11(7):1250–8. doi: 10.1016/j.hrthm.2014.02.

008 PMID: 24560692

8. Allessie M, Ausma J, Schotten U. Electrical, contractile and structural remodeling during atrial fibrilla-

tion. Cardiovasc Res. 2002; 54(2):230–46. PubMed PMID: 12062329. PMID: 12062329

9. Pandozi C, Bianconi L, Villani M, Gentilucci G, Castro A, Altamura G, et al. Electrophysiological charac-

teristics of the human atria after cardioversion of persistent atrial fibrillation. Circulation. 1998; 98

(25):2860–5. PubMed PMID: 9860788. PMID: 9860788

10. Ausma J, Wijffels M, Thone F, Wouters L, Allessie M, Borgers M. Structural changes of atrial myocar-

dium due to sustained atrial fibrillation in the goat. Circulation. 1997; 96(9):3157–63. PubMed PMID:

9386188. PMID: 9386188

11. Frustaci A, Chimenti C, Bellocci F, Morgante E, Russo MA, Maseri A. Histological substrate of atrial

biopsies in patients with lone atrial fibrillation. Circulation. 1997; 96(4):1180–4. Epub 1997/08/19.

PubMed PMID: 9286947. PMID: 9286947

12. Aldhoon B, Melenovsky V, Peichl P, Kautzner J. New insights into mechanisms of atrial fibrillation. Phy-

siol Res. 2010; 59(1):1–12. PubMed PMID: 19249911. PMID: 19249911

13. Burstein B, Nattel S. Atrial fibrosis: mechanisms and clinical relevance in atrial fibrillation. J Am Coll Car-

diol. 2008; 51(8):802–9. doi: 10.1016/j.jacc.2007.09.064 PMID: 18294563

14. van Brakel TJ, van der Krieken T, Westra SW, van der Laak JA, Smeets JL, van Swieten HA. Fibrosis

and electrophysiological characteristics of the atrial appendage in patients with atrial fibrillation and

structural heart disease. J Interv Card Electrophysiol. 2013; 38(2):85–93. doi: 10.1007/s10840-013-

9820-8 PMID: 24026967

15. Nguyen BL, Fishbein MC, Chen LS, Chen PS, Masroor S. Histopathological substrate for chronic atrial

fibrillation in humans. Heart Rhythm. 2009; 6(4):454–60. PubMed Central PMCID: PMCPMC2662134.

doi: 10.1016/j.hrthm.2009.01.010 PMID: 19324302

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 11 / 13

16. Wu H, Xie J, Li GN, Chen QH, Li R, Zhang XL, et al. Possible involvement of TGF-beta/periostin in fibro-

sis of right atrial appendages in patients with atrial fibrillation. Int J Clin Exp Pathol. 2015; 8(6):6859–69.

PubMed PMID: 26261573; PubMed Central PMCID: PMCPMC4525907. PMID: 26261573

17. Corradi D, Callegari S, Benussi S, Nascimbene S, Pastori P, Calvi S, et al. Regional left atrial interstitial

remodeling in patients with chronic atrial fibrillation undergoing mitral-valve surgery. Virchows Arch.

2004; 445(5):498–505. doi: 10.1007/s00428-004-1040-2 PMID: 15221371

18. Guo Y, Lip GY, Apostolakis S. Inflammation in atrial fibrillation. J Am Coll Cardiol. 2012; 60(22):2263–

70. doi: 10.1016/j.jacc.2012.04.063 PMID: 23194937

19. Boos CJ, Anderson RA, Lip GY. Is atrial fibrillation an inflammatory disorder? Eur Heart J. 2006; 27

(2):136–49. doi: 10.1093/eurheartj/ehi645 PMID: 16278230

20. Verheule S, Wilson E, Everett Tt, Shanbhag S, Golden C, Olgin J. Alterations in atrial electrophysiology

and tissue structure in a canine model of chronic atrial dilatation due to mitral regurgitation. Circulation.

2003; 107(20):2615–22. Epub 2003/05/07. PubMed Central PMCID: PMC1995672. doi: 10.1161/01.

CIR.0000066915.15187.51 PMID: 12732604

21. Liao CH, Akazawa H, Tamagawa M, Ito K, Yasuda N, Kudo Y, et al. Cardiac mast cells cause atrial fibril-

lation through PDGF-A-mediated fibrosis in pressure-overloaded mouse hearts. J Clin Invest. 2010;

120(1):242–53. Epub 2009/12/30. PubMed Central PMCID: PMC2798688. doi: 10.1172/JCI39942

PMID: 20038802

22. Nakamura Y, Nakamura K, Fukushima-Kusano K, Ohta K, Matsubara H, Hamuro T, et al. Tissue factor

expression in atrial endothelia associated with nonvalvular atrial fibrillation: possible involvement in

intracardiac thrombogenesis. Thromb Res. 2003; 111(3):137–42. Epub 2003/12/18. PMID: 14678810

23. Chen MC, Chang JP, Liu WH, Yang CH, Chen YL, Tsai TH, et al. Increased inflammatory cell infiltration

in the atrial myocardium of patients with atrial fibrillation. Am J Cardiol. 2008; 102(7):861–5. Epub 2008/

09/23. doi: 10.1016/j.amjcard.2008.05.038 PMID: 18805111

24. Yamashita T, Sekiguchi A, Iwasaki YK, Date T, Sagara K, Tanabe H, et al. Recruitment of immune cells

across atrial endocardium in human atrial fibrillation. Circ J. 2010; 74(2):262–70. Epub 2009/12/17.

PMID: 20009387

25. Platonov PG, Mitrofanova LB, Orshanskaya V, Ho SY. Structural abnormalities in atrial walls are associ-

ated with presence and persistency of atrial fibrillation but not with age. J Am Coll Cardiol. 2011; 58

(21):2225–32. Epub 2011/11/15. doi: 10.1016/j.jacc.2011.05.061 PMID: 22078429

26. Rudolph V, Andrie RP, Rudolph TK, Friedrichs K, Klinke A, Hirsch-Hoffmann B, et al. Myeloperoxidase

acts as a profibrotic mediator of atrial fibrillation. Nat Med. 2010; 16(4):470–4. PubMed Central PMCID:

PMCPMC2880896. doi: 10.1038/nm.2124 PMID: 20305660

27. Smorodinova N, Lantova L, Blaha M, Melenovsky V, Hanzelka J, Pirk J, et al. Bioptic Study of Left and

Right Atrial Interstitium in Cardiac Patients with and without Atrial Fibrillation: Interatrial but Not Rhythm-

Based Differences. PLoS One. 2015; 10(6):e0129124. PubMed Central PMCID: PMCPMC4466374.

doi: 10.1371/journal.pone.0129124 PMID: 26067062

28. Yamashita T, Sekiguchi A, Suzuki S, Ohtsuka T, Sagara K, Tanabe H, et al. Enlargement of the left

atrium is associated with increased infiltration of immune cells in patients with atrial fibrillation who had

undergone surgery. J Arrhythm. 2015; 31(2):78–82. PubMed Central PMCID: PMCPMC4550127. doi:

10.1016/j.joa.2014.07.003 PMID: 26336536

29. Begieneman MP, Rijvers L, Kubat B, Paulus WJ, Vonk AB, van Rossum AC, et al. Atrial fibrillation coin-

cides with the advanced glycation end product N(epsilon)-(carboxymethyl)lysine in the atrium. Am J

Pathol. 2015; 185(8):2096–104. doi: 10.1016/j.ajpath.2015.04.018 PMID: 26216282

30. Caforio AL, Pankuweit S, Arbustini E, Basso C, Gimeno-Blanes J, Felix SB, et al. Current state of knowl-

edge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the

European Society of Cardiology Working Group on Myocardial and Pericardial Diseases. Eur Heart J.

2013; 34(33):2636–48, 48a-48d. doi: 10.1093/eurheartj/eht210 PMID: 23824828

31. Maisch B, Richter A, Sandmoller A, Portig I, Pankuweit S, Network BM-HF. Inflammatory dilated cardio-

myopathy (DCMI). Herz. 2005; 30(6):535–44. doi: 10.1007/s00059-005-2730-5 PMID: 16170686

32. Aldhoon B, Kucera T, Smorodinova N, Martinek J, Melenovsky V, Kautzner J. Associations between

cardiac fibrosis and permanent atrial fibrillation in advanced heart failure. Physiol Res. 2013; 62(3):247–

55. PMID: 23489193

33. De Jong AM, Maass AH, Oberdorf-Maass SU, Van Veldhuisen DJ, Van Gilst WH, Van Gelder IC. Mech-

anisms of atrial structural changes caused by stretch occurring before and during early atrial fibrillation.

Cardiovasc Res. 2011; 89(4):754–65. doi: 10.1093/cvr/cvq357 PMID: 21075756

34. Qu YC, Du YM, Wu SL, Chen QX, Wu HL, Zhou SF. Activated nuclear factor-kappaB and increased

tumor necrosis factor-alpha in atrial tissue of atrial fibrillation. Scand Cardiovasc J. 2009; 43(5):292–7.

doi: 10.1080/14017430802651803 PMID: 19169931

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 12 / 13

35. Haemers P, Hamdi H, Guedj K, Suffee N, Farahmand P, Popovic N, et al. Atrial fibrillation is associated

with the fibrotic remodelling of adipose tissue in the subepicardium of human and sheep atria. Eur Heart

J. 2015.

36. de Oliveira IM, Oliveira BD, Scanavacca MI, Gutierrez PS. Fibrosis, myocardial crossings, disconnec-

tions, abrupt turns, and epicardial reflections: do they play an actual role in human permanent atrial fibril-

lation? A controlled necropsy study. Cardiovasc Pathol. 2013; 22(1):65–9. doi: 10.1016/j.carpath.2012.

06.001 PMID: 22917538

37. Rocken C, Peters B, Juenemann G, Saeger W, Klein HU, Huth C, et al. Atrial amyloidosis: an arrhyth-

mogenic substrate for persistent atrial fibrillation. Circulation. 2002; 106(16):2091–7. PMID: 12379579

38. Afanasyeva M, Georgakopoulos D, Rose NR. Autoimmune myocarditis: cellular mediators of cardiac

dysfunction. Autoimmun Rev. 2004; 3(7–8):476–86. doi: 10.1016/j.autrev.2004.08.009 PMID:

15546794

39. Marty RR, Eriksson U. Dendritic cells and autoimmune heart failure. Int J Cardiol. 2006; 112(1):34–9.

doi: 10.1016/j.ijcard.2006.06.022 PMID: 16891018

40. Pistulli R, Konig S, Drobnik S, Kretzschmar D, Rohm I, Lichtenauer M, et al. Decrease in dendritic cells

in endomyocardial biopsies of human dilated cardiomyopathy. Eur J Heart Fail. 2013; 15(9):974–85.

doi: 10.1093/eurjhf/hft054 PMID: 23603088

41. Begieneman MP, Emmens RW, Rijvers L, Kubat B, Paulus WJ, Vonk AB, et al. Ventricular myocarditis

coincides with atrial myocarditis in patients. Cardiovasc Pathol. 2016; 25(2):141–8. doi: 10.1016/j.

carpath.2015.12.001 PMID: 26764148

Immune cells in the atrial myocardium and atrial fibrillation

PLOS ONE | DOI:10.1371/journal.pone.0172691 February 22, 2017 13 / 13